Expansion of hematopoietic cells using midkine or pleiotrophin

Abstract

The present invention provides novel use of the MK family that is used alone as an agent for proliferating hematopoietic stem cells and hematopoietic precursor cells. The invention also provides an agent for remarkably enhancing the above-described effect for promoting the proliferation of hematopoietic stem cells and hematopoietic precursor cells, comprising the MK family in combination with known hematopoietic factors such as IL-3, IL-6, G-CSF, GM-CSF, M-CSF, SCF, and EPO.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional application of U.S. application Ser. No. 09 / 214,569, now U.S. Pat. No. 6,383,480, which is a National Phase Application, filed Jan. 7, 1999, under 35 U.S.C §371, of International Application No. PCT / JP97 / 02401, filed Jul. 10, 1997.TECHNICAL FIELD[0002]This invention relates to a novel use of MK to promote proliferation and differentiation of hematopoietic stem cells and hematopoietic precursor cells in hematopoietic tissues, peripheral blood, or umbilical cord blood synergistically with other hematopoietic factors.BACKGROUND ART[0003]In blood, there exist various hemocytes having different shapes and functions, including erythrocytes, leukocytes, and platelets, which play important roles in maintaining homeostatis of the living body. These mature hemocytes have their own life-spans. For maintaining the hemocyte count at a constant level, hemocytes must be incessantly produced to make up for the number of h...

AI Technical Summary

Benefits of technology

[0012]The present inventors have found that single use of a novel growth factor called midkine (MK) or pleiotrophin (PTN) promotes the proliferation and differentiation of hematopoietic stem cells and hematopoietic precursor cells (also called hematopoietic cells) of mammals such as mice and humans in vitro. MK also exerts an extremely remarkable synergistic effect for proliferating and differentiating hematopoietic cells when used together with SCF, M-CSF, G-CSF, GM-CSF, IL-3, and IL-6. Furthermore, the inventors have found that MK or PTN promotes rapid recovery of neutrophils in neutropenia of mammals.

[0019]In the colony assay for human peripheral blood mononuclear cells in the presence of various cytokines, as shown in FIG. 1, colonies were not formed at all in the system without cytokines, but formed in the system supplemented with MK similarly as with GM-CSF and IL-3. The colony size tends to be larger in the system with MK added than in the systems with cytokines such as G-CSF, GM-CSF, and IL-3 added. These results indicate that MK alone has the activity to maintain the viability of human peripheral blood stem cells or hematopoietic precursor cells, or promote their proliferation. Furthermore, the combined use of MK with other cytokines such as M-CSF, G-CSF, GM-CSF, IL-3, and IL-6 synergistically promotes colony-forming capability. For example, the number of colonies increase 7 to 9-fold in the cases of combined use of MK with G-CSF, GM-CSF, or IL-3 as compared with those of the single use of MK, G-CSF, GM-CSF, or IL-3. Also, the combination of MK+G-CSF+IL-3+IL-6 remarkably increased the number of colonies formed as compared with that of GM-CSF+IL-3+IL-6, and the combination of MK+G-CSF+IL-6 significantly increased the number of colonies as compared with that of G-CSF+IL-6. In another experiment using a source of human peripheral blood mononuclear cells different from that used for the experiments described in FIG. 1, treatment with MK, GM-CSF, or IL-3 alone produced primarily GM colonies, while use of G-CSF alone produced primarily G colonies. Combinations of MK+G-CSF+GM-CSF, or of MK+G-CSF significantly increased the number of G colonies as compared with the use of G-CSF alone. That is, MK is considered to synergistically promote the proliferation, differentiation and maturation of CFU-GM of G-CSF, increasing the number of neutrophils in the peripheral blood.

[0025]Results of the colony assay for mouse spleen cells in the system of the M3434 medium supplemented with MK are shown in FIGS. 4 and 5. FIG. 4 illustrates the number of CFU-GM colonies or CFU-G colonies. Although CFU-GM colonies or CFU-G colonies can be formed with the M3434 medium alone, the number of colonies generally increases in the system supplemented with the MK as compared with the M3434 medium alone. Especially, when MK is added at the concentration of 1 to 10 ng / ml, the colony number remarkably increased 2 to 3-fold on the 8th and 10th day of the assay. FIG. 5 illustrates the number of colony-forming unit-mixed (CFU-Mix). CFU-Mix are multipotential stem cells at a slightly differentiated stage, having lower self-renewal capability than blast colony-forming units (CFU-BL) which are the most undifferentiated cells identifiable by the in vitro colony assay and are thought to contain cells capable of differentiating to erythrocytes, leukocytes, and platelets. In the system supplemented with MK, CFU-Mix colony significantly increased in number. That is, MK, at least by its combined use with IL-3, IL-6, SCF and EPO, is thought to significantly promote the proliferation and differentiation of hematopoietic stem cells or immature hematopoletic cells close to them. These activities are thought to be very useful for the proliferation of hematopoietic stem cells in vitro for the transplantation of bone marrow and peripheral blood stem cells, or gene transfer to hematopoietic stem cells.

[0031]Next, using a hematopoietic stem cell assay medium complete type (Lot No. 96101601; Kyokuto Seiyaku Kogyo) containing IL-3, SCF, G-CSF, and EPO, the colony assay was carried out with peripheral blood cells from a healthy normal individual. This assay is considered to be performed under conditions closer to in vivo. Results with MK are shown in FIGS. 12 and 13, and those with PTN in FIGS. 14 and 15. No increase in BFU-E was observed with any combinations including MK+IL-3, MK+SCF, and MK+G-CSF. However, the combinations of MK+EPO and PTN+EPO were assumed to increase BFU-E. Erythroblast precursor cells, BFU-E, were formed on the 14th day of culture, and are known to be more undifferentiated than CFU-E formed on the 5th to 7th days. Addition of MK or PTN to a Kyokuto complete medium resulted in formation of at the highest 2 or more times as many BFU-E as the complete medium alone on the 12th day after the initiation of culture. These results indicate that at least the addition of MK to the complete medium results in promoting the proliferation of erythroblasts as well. As described above, it is evident that the MK family is capable of acting on hematopoietic stem cells and hematopoietic precursor cells in the hematopoietic tissues of mammals to maintain, proliferate, and differentiate them, and synergistically or additionally enhancing the above-described functions by the combined use with various cytokines such as SCF, M-CSF, G-CSF, GM-CSF, IL-3 AND IL-6. Especially, the MK family remarkably promotes the proliferation of CFU-Mix which is very close to multipotential stem cells, under conditions closer to in vivo. The MK family also promotes the proliferation and differentiation of granulocyte / macrophage precursor cells and exerts the remarkable neutrophil increasing effect in an in vivo neutropenia model. This MK family alone or in combination with more than one cytokine including SCF, M-CSF, G-CSF, GM-CSF, IL-3, and IL-6 can be clinically applied and, especially, used for the ex vivo expansion of hematopoietic stem cells in the transplantation of bone marrow and stem cells derived from the peripheral blood and umbilical cord blood. In addition, the MK family is expected to be used for the treatment of patients with and prevention of neutropenia, refractory anemia, and leukemia caused by cancer chemotherapy. Furthermore, the MK family would be used in the future for proliferating stem cells for gene therapy targeting hematopoietic stem cells; Especially, it is very promising to increase the dose density in cancer chemotherapy by the combined use of MK with G-CSF, improving effects of chemotherapy by increasing the dose of antitumor drugs or shortening the administration period.

Problems solved by technology

However, even now, human hematopoietic stem cells have been neither isolated nor clarified as to what extent they can repeat self-renewal.

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